The ESCRT-II Subunit EAP20/VPS25 and the Bro1 Domain Proteins HD-PTP and BROX Are Individually Dispensable for Herpes Simplex Virus 1 Replication
The ESCRT-II Subunit EAP20/VPS25 and the Bro1 Domain Proteins HD-PTP and BROX Are Individually Dispensable for Herpes Simplex Virus 1 Replication
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DOI：
10.1128/jvi.01641-19
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发表时间：
2020-02-01
影响因子：
5.4
通讯作者：
Wilson, Duncan W.
中科院分区：
文献类型：
--
作者：
Barnes, Jenna;Wilson, Duncan W.
嗜神经性疱疹病毒单纯疱疹病毒1型（HSV-1）和伪狂犬病病毒（PRV）在感染细胞质中组装期间的衣壳破坏被认为涉及晚期作用细胞ESCRT（转运所需的内体分选复合物）组分ESCRT-III和VPS 4（液泡蛋白分选4）。然而，与许多其他包膜病毒家族的成员不同，HSV-1似乎不需要ESCRT-I亚基TSG 101或含有Bro 1结构域的蛋白阿利克斯（Alg-2相互作用蛋白X）来招募和激活ESCRT-III。已知能够调节ESCRT-III功能的替代细胞因子包括ESCRT-II复合物和Bro 1家族的其他成员。因此，我们使用小干扰RNA（siRNA）敲除必需的ESCRT-II亚基EAP 20/VPS 25（ELL-associated protein 20/vacuolar protein sorting 25）和Bro 1蛋白HD-PTP（含His结构域的蛋白酪氨酸磷酸酶）和BROX（含Bro 1结构域和CAAX基序）。我们通过Western印迹证明了靶蛋白水平的降低，并使用定量显微镜测定来确认ESCRT-II和HD-PTP功能的丧失。我们发现，在单步复制实验中，HSV-1的最终产量在EAP 20、HD-PTP或BROX缺失后保持不变。重要提示HSV-1是人类神经系统的病原体，它使用自身的病毒编码蛋白和正常细胞ESCRT机制来驱动其包膜的构建。HSV-1结构蛋白如何与ESCRT组分相互作用以及病毒利用细胞ESCRT蛋白的哪些子集仍然是未知的。在这里，我们证明了ESCRT-II复合物的一个重要组成部分和两个ESCRT相关的Bro 1蛋白是HSV-1复制所必需的。
Capsid envelopment during assembly of the neurotropic herpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) in the infected cell cytoplasm is thought to involve the late-acting cellular ESCRT (endosomal sorting complex required for transport) components ESCRT-III and VPS4 (vacuolar protein sorting 4). However, HSV-1, unlike members of many other families of enveloped viruses, does not appear to require the ESCRT-I subunit TSG101 or the Bro1 domain-containing protein ALIX (Alg-2-interacting protein X) to recruit and activate ESCRT-III. Alternative cellular factors that are known to be capable of regulating ESCRT-III function include the ESCRT-II complex and other members of the Bro1 family. We therefore used small interfering RNA (siRNA) to knock down the essential ESCRT-II subunit EAP20/VPS25 (ELL-associated protein 20/vacuolar protein sorting 25) and the Bro1 proteins HD-PTP (His domain-containing protein tyrosine phosphatase) and BROX (Bro1 domain and CAAX motif containing). We demonstrated reductions in levels of the targeted proteins by Western blotting and used quantitative microscopic assays to confirm loss of ESCRT-II and HD-PTP function. We found that in single-step replication experiments, the final yields of HSV-1 were unchanged following loss of EAP20, HD-PTP, or BROX.IMPORTANCE HSV-1 is a pathogen of the human nervous system that uses its own virus-encoded proteins and the normal cellular ESCRT machinery to drive the construction of its envelope. How HSV-1 structural proteins interact with ESCRT components and which subsets of cellular ESCRT proteins are utilized by the virus remain largely unknown. Here, we demonstrate that an essential component of the ESCRT-II complex and two ESCRT-associated Bro1 proteins are dispensable for HSV-1 replication.